Transcriptional Regulation by Hypoxia-Inducible Factors in Tumor Cells

Abstract: Cancer is a major cause of human morbidity and mortality, and the risk of developing cancer is about one in three life times. Neuroblastoma is the most common extra-cranial solid tumor among children and arises from early sympathetic nervous system (SNS) cells arrested in their development. Generally, a low tumor cell differentiation correlates to poor prognosis. Solid tumors, like neuroblastoma, frequently contain regions of oxygen deficiency ? hypoxia ? caused by a high rate of cellular proliferation and abnormal intratumoral blood supply. In this hypoxic microenvironment cancer cells undergo genetic and molecular changes, allowing continued survival and proliferation. Tumor hypoxia is also associated with increased aggressiveness, resistance to therapy and poor outcome. Cancer cells become less differentiated in response to hypoxia, which we previously demonstrated in neuroblastoma as well as breast cancer cells, indicating an evolvement of a more aggressive phenotype. In the present studies we find evidence of potential mechanisms behind the hypoxia-mediated de-differentiation of neuroblastoma cells. Hypoxia (1% O2) induced the expression of the negative transcription factor ID2 (Paper I), involved in blocking the function of tissue-specific basic helix-loop-helix (bHLH) proteins, such as the SNS-specifying transcription factors HASH-1 and dHAND. Hypoxic up-regulation of ID2 was dependent on direct in vivo DNA-binding and activity of hypoxia-inducible factors (HIF), the master transcriptional regulators of oxygen homeostasis. Induction of ID2 expression occurs as an early HIF-mediated hypoxic event, potentially leading to a more immature state. HIF-1alpha and HIF-2alpha, however differently, are both essential for normal development and are highly implicated in tumor progression. In Paper II we show that HIF-1alpha and HIF-2alpha share several target genes, but mediate regulation of these under different temporal and oxygen-dependent conditions. Interestingly, HIF-2alpha, but not HIF-1alpha, was present in neuroblastoma tumor cells near blood vessels, and thus in apparently better oxygenized tumor regions. In vitro, HIF-1alpha protein was transiently stabilized at hypoxia and primarily governed acute hypoxic responses, whereas HIF-2alpha became more important at prolonged hypoxia. In addition, high HIF-2alpha activity, including induction of classic hypoxic targets such as VEGF, was detected in cultured neuroblastoma cells already at 5% O2, a physiologically relevant oxygen level, similar to the findings in vivo. In a large clinical neuroblastoma material, significant correlations between high HIF-2alpha levels and high VEGF content, advanced tumor stage and poor outcome were found. These observations clearly suggest an oncogenic role of HIF-2alpha, and implicate HIF-2alpha as an independent prognostic marker in neuroblastoma. The MXI1 (MAX-interactor 1) gene, a reported MYC antagonist, has been detected by us and others as a commonly hypoxia-induced gene. In Paper III we further demonstrate that HIF proteins, via direct binding to hypoxia-response elements (HRE), up-regulate MXI1 mRNA and protein in both hypoxic neuroblastoma and breast cancer cells. Interestingly, reducing MXI1 levels had no overall effects on MYC/MYCN activity in hypoxic neuroblastoma cells. Instead, MXI1 appeared to be important in augmenting the hypoxic response, potentially by enhancing specific HIF-1 target gene induction. HIF proteins are primarily stabilized and activated in response to lowered oxygen concentrations. However, growth factor-induced signaling can promote HIF-1alpha protein synthesis as well as transactivation, even under normoxic conditions. In Paper IV we characterize a novel such a pathway, where stem cell factor (SCF)-evoked c-Kit-signaling leads to increased HIF-1alpha protein, HRE-activation and induction of several HIF-1alpha targets, such as VEGF and GLUT1, already at normoxia. In addition we find a reciprocal positive feedback loop between c-Kit and HIF-1alpha, where induced HIF-1alpha mediates reinforcement of c-Kit expression. Overall, this thesis shows the impact of HIF proteins on tumor cell behavior, principally as central hypoxic transcriptional regulators governing the expression of genes with potential importance in several biological processes, such as growth and differentiation, determining cancer cell aggressiveness as well as adaptation to low oxygen conditions.